1. Field
The present disclosure relates to methods of determining a ratio of RNA species in a sample by amplifying target RNA in a sample into DNA without a bias.
2. Description of the Related Art
Transcriptome analysis is an important approach to diagnose a disease, detect an underlying cause of a disease, and/or find new targets for drug development. In the transcriptome analysis, a small amount of RNA obtained from a sample needs to be amplified.
RNA expressed from a cell or a virus has a structure with intrinsic activity. For example, mRNA of a eukaryotic cell has a 5′-cap structure and a 3′-poly (adenylate) sequence. However, these groups may be removed or deformed for a least a portion of the RNA while a biological sample is stored or analyzed. For example, RNA isolated from a formalin-fixed paraffin-embedded (FFPE) tissue that is frozen or stored at room temperature has a length of about 300 bp or less, indicating degradation of RNA in the sample. The FFPE method is widely used to preserve samples of patients' tissues at room temperature because it is a simple and inexpensive method. FFPE samples of many patients which are preserved for a long period of time provide an invaluable resource for the retrospective study of disease. However, most RNA isolated from a FFPE tissue are fragmented, making it difficult to analyze by conventional methods.
RNA is generally analyzed by reverse transcribing the RNA to produce cDNA, amplifying the cDNA, and assaying the amplified product. However, in such a method, the fragmented RNA may not be converted into cDNA and is, thus, effectively lost when cDNA is synthesized and amplified. This results in a polarization in the analysis of the RNA content of the sample, with certain RNA “species” being underrepresented by the amplified DNA.
Thus, there is a need to develop a method of producing DNA whereby the level of RNA species in a sample is maintained, i.e., the level of RNA species is not polarized. In addition, there is a need to develop a method of determining a ratio of RNA species in a sample in an efficient way.